Whole Blood Bacterial DNA Isolation Kit

Whole Blood Bacterial DNA Isolation Kit Instructions

Product Composition

Product Storage and Expiry Date

1．  Please store lysozyme at 2~8℃.

2．  If other reagents and items are stored at normal temperature (0~30℃), their performance will remain unchanged within two years; if the product is stored at 2~8℃, the validity period of the product can be extended to more than two years (2~8℃ Stored products should be returned to room temperature before use).

Product Description

This product is suitable for rapid separation and purification of total blood DNA from 350 µl of fresh or frozen human or animal whole blood. After lysozyme breaks the bacteria in the whole blood, the lysis solution dissolves the whole blood and the bacteria in the blood, and then precipitates with Buffer L2 to remove the hemoglobin. The bacterial DNA in the supernatant is adsorbed to the purification column together with the cell DNA, and passed through Buffer WA After washing with Buffer WB to remove remaining proteins and PCR inhibitors on the membrane, the DNA is eluted with Buffer TE and can be used immediately for various molecular biology experiments.

Reagents and items that users need to prepare by themselves

3．  Deionized pure water, absolute ethanol

4．  1.5 ml centrifuge tube

5．  Pipette tips (to avoid contamination between samples, it is recommended to use pipette tips with filters)

6．  Disposable gloves and protective equipment and tissues

7．  Desktop small volume centrifuge (can be equipped with a rotor for centrifuging 1.5 ml centrifuge tubes and 2 ml centrifuge tubes)

8．  Water bath or dry bath, vortex oscillator

Preparation before use

1.        If the centrifuge has a refrigeration function, please set the temperature to 25°C.

2.        Set the water bath temperature to 37 °C and incubate Buffer TE to 37°C.

3.        Prepare an appropriate amount of 100 mg/ml lysozyme solution based on the number of specimens to be extracted at one time (calculated as 50 µl of lysozyme solution is required for each specimen): For example, if you want to extract bacterial genomic DNA from 10 specimens, weigh 55 mg of lysozyme. Dry powder, add 550 µl deionized pure water to prepare 550 µl lysozyme solution.

Note: Repeated freezing and thawing of lysozyme solution has a great impact on its activity. If a large amount of lysozyme solution is prepared at one time, it should be divided into small portions and stored at -20°C. If there is any remaining lysozyme solution after thawing and use, it should be Discard and do not freeze again.

4.        Add absolute ethanol to Buffer WA and Buffer WB according to the instructions on the label of the reagent bottle, and check the box on the label to mark "Ethanol has been added".

Steps:

Note: The dosage of Buffer L1 and Buffer L2 must be accurately operated according to the volume ratio of Buffer L1: the volume of blood after lysozyme treatment: Buffer L2=300 µl:400 µl:300 µl, otherwise the subsequent steps will not be carried out.

1.       Add 50 µl of freshly prepared lysozyme solution to a 1.5 ml centrifuge tube, then add 350 µl of whole blood, mix evenly, and place in a 37°C water bath for 30 minutes.

2.       Add 300 µl Buffer L1, cover the tube, shake vigorously 3-5 times, and vortex for 30 seconds.

* If DNA is extracted from fresh blood, RNA in the blood may be separated and purified at the same time, but the presence of RNA does not affect PCR-related experiments. If you want to remove RNA contamination, you can add 4 µl RNase A (100 mg/ml, not provided with this kit) in this step.

3.       Add 300 µl Buffer L2, cover the tube, shake the centrifuge tube vigorously 3-5 times, and vortex for 30 seconds to mix.

* A large amount of hemoglobin will precipitate during this step.

4.       Centrifuge at 13,000 rpm for 2 minutes.

5.       Pour the supernatant in step 4 into the nucleic acid purification column (place the nucleic acid purification column in a 2 ml centrifuge tube), cover the tube, and centrifuge at 12,000 rpm for 30 seconds.

* When DNA is extracted from the blood of some animals, due to less hemoglobin, the volume of the supernatant obtained by centrifugation may be larger than the volume of the purification column . In this case, it is recommended to pipet 700 µl of the supernatant into the nucleic acid purification column, or transfer the supernatant Perform this step in two batches.

* If hemoglobin remains on the purification column membrane, it is normal and can be washed away with Buffer WA.

6.       Discard the filtrate in the 2 ml centrifuge tube, place the nucleic acid purification column back into the 2 ml centrifuge tube, add 500 µl Buffer WA to the nucleic acid purification column, cap the tube, and centrifuge at 12,000 rpm for 30 seconds.

* There is no need to discard the filtrate completely. If you want to avoid contamination of the centrifuge by the filtrate adhering to the mouth of the centrifuge tube, place the 2 ml centrifuge tube upside down on a paper towel and tap it once.

* Confirm that absolute ethanol has been added to Buffer WA.

7.       Discard the filtrate in the 2 ml centrifuge tube, place the nucleic acid purification column back into the 2 ml centrifuge tube, add 600 µl Buffer WB to the nucleic acid purification column, cap the tube, and centrifuge at 12,000 rpm for 30 seconds.

* Confirm that absolute ethanol has been added to Buffer WB.

8.       Discard the filtrate in the 2 ml centrifuge tube, place the nucleic acid purification column back into the 2 ml centrifuge tube, and centrifuge at 14,000 rpm for 1 minute.

* If the centrifuge speed cannot reach 14000 rpm, centrifuge at the highest speed for 2 minutes.

* Do not omit this step, otherwise the purified nucleic acid may be mixed with ethanol, which may affect subsequent PCR results.

9.       Discard the 2 ml centrifuge tube, place the nucleic acid purification column in a clean 1.5 ml centrifuge tube, add 60 to 100 µl Buffer TE incubated at 37°C in the center of the purification column, cover the tube cap, and let stand at room temperature for 1 minute, 12000 Centrifuge at rpm for 30 seconds.

* If the centrifuge does not have a leak-proof lid, please change the centrifugation conditions to 8000 rpm for 1 minute to prevent the tube lid from falling off and damaging the centrifuge.

10.    Discard the purification column, and the eluted DNA can be used immediately for various molecular biology experiments; or the DNA can be stored at -20°C for later use.